|
| Status |
Public on Jan 08, 2016 |
| Title |
# 2VHL/HIF2A Vhlh fl/fl w/ Adeno-Cre-GFP Cy5 and # 2VHL/HIF2A Vhlh fl/fl w/ Adeno-GFP. Control Cy3 |
| Sample type |
RNA |
| |
|
| Channel 1 |
| Source name |
Animal 2; Primary kidney epithelial cells from Vhlh flox/flox, Hif2a flox/flox animal. Cells treated with Adeno-GFP virus. Control cell culture Cy3 labeled.
|
| Organism |
Mus musculus |
| Characteristics |
Sex: female
cell type: primary kidney epithelial cell
age: 50 days (adult)
|
| Treatment protocol |
After 5–6 days in culture, cells were infected with adenoviruses expressing GFP (Vector Biolabs, 1060) or Cre-GFP (Vector Biolabs, 1700).
|
| Growth protocol |
Kidneys were dissected from 2-month-old floxed mice. After removing the capsule under sterile conditions, kidneys were mashed with a razor blade on ice and digested in collagenase II (Gibco) and soya trypsin inhibitor (Gibco) solution at 37°C for 30 min. The cell suspension was filtered through a 70 µm cell strainer and washed in HBSS + 5% FCS. Erythrocytes were lysed for 1 min using standard ACK buffer. Cells were resuspended in complete K-1 culture medium [Dulbecco's modified Eagle's medium (DMEM):Hams F12] (50:50), supplemented with 0.5% foetal calf serum, hormone mix [5 µg/ml insulin, 1.25 ng/ml prostaglandin E1 (PGE1), 34 pg/ml triiodothyronine, 5 µg/ml Apo-transferrin, 1.73 ng/ml sodium selenite and 18 ng/ml of hydrocortisone] and 25 ng/ml epidermal growth factor (EGF). Cells were counted and seeded at a density of 1 × 106 cells on standard 100 mm plastic tissue culture plates.
|
| Extracted molecule |
total RNA |
| Extraction protocol |
Machery Nagel Nucleospin RNA II according to producer instructions.
|
| Label |
Cy3
|
| Label protocol |
standard Agilent protocol
|
| |
|
| Channel 2 |
| Source name |
Animal 2. Primary kidney epithelial cells from Vhlh flox/flox, Hif2a flox/flox animals. Cells treated with Adeno-Cre-GFP virus labeled with Cy5.
|
| Organism |
Mus musculus |
| Characteristics |
Sex: female
cell type: primary kidney epithelial cell
age: 50 days (adult)
|
| Treatment protocol |
After 5–6 days in culture, cells were infected with adenoviruses expressing GFP (Vector Biolabs, 1060) or Cre-GFP (Vector Biolabs, 1700).
|
| Growth protocol |
Kidneys were dissected from 2-month-old floxed mice. After removing the capsule under sterile conditions, kidneys were mashed with a razor blade on ice and digested in collagenase II (Gibco) and soya trypsin inhibitor (Gibco) solution at 37°C for 30 min. The cell suspension was filtered through a 70 µm cell strainer and washed in HBSS + 5% FCS. Erythrocytes were lysed for 1 min using standard ACK buffer. Cells were resuspended in complete K-1 culture medium [Dulbecco's modified Eagle's medium (DMEM):Hams F12] (50:50), supplemented with 0.5% foetal calf serum, hormone mix [5 µg/ml insulin, 1.25 ng/ml prostaglandin E1 (PGE1), 34 pg/ml triiodothyronine, 5 µg/ml Apo-transferrin, 1.73 ng/ml sodium selenite and 18 ng/ml of hydrocortisone] and 25 ng/ml epidermal growth factor (EGF). Cells were counted and seeded at a density of 1 × 106 cells on standard 100 mm plastic tissue culture plates.
|
| Extracted molecule |
total RNA |
| Extraction protocol |
Machery Nagel Nucleospin RNA II according to producer instructions.
|
| Label |
Cy5
|
| Label protocol |
standard Agilent protocol
|
| |
|
| |
| Hybridization protocol |
standard Agilent protocol
|
| Scan protocol |
standard Agilent protocol
|
| Description |
One of the biological replicates avaraged to obtain values for Vhlh-/-, Hif2a-/-_logRatio
mouse number 2VHL/HIF2A_primary kidney epithelial cells from Vhlh flox/flox infected with Adeno-Cre-GFP virus Cy5 labeled and mouse number_ 2VHL/HIF2A_primary kidney epithelial cells from Vhlh flox/flox infected with Adeno-GFP virus. Control cell culture Cy3 labeled.
|
| Data processing |
Data were processed using R Bioconductor (limma). For the final analysis the gene expression levels were dye effect corrected and values for biological replicates were averaged to obtain a matrix with gene expression levels for 4 independent biological conditions (Vhlh-/-, Vhlh-/-/Hif1α-/-, Vhlh-/-/Hif2α-/- and Vhlh-/-/Hif1α-/-/Hif2α-/-).
normalized log2 ratio (Cy5/Cy3) representing values for primary kidney cells with defined alleles floxed infected either with CRE-GFP adenovirus (excision) or GFP adenovirus (control).
|
| |
|
| Submission date |
Dec 07, 2015 |
| Last update date |
Jan 09, 2016 |
| Contact name |
Michal Rajski |
| E-mail(s) |
rajski.michal@gmail.com
|
| Organization name |
University of Zurich
|
| Department |
Institute of Physiology
|
| Lab |
23K86
|
| Street address |
Winterthurerstrasse 190
|
| City |
Zurich |
| ZIP/Postal code |
CH - 8057 |
| Country |
Switzerland |
| |
|
| Platform ID |
GPL10333 |
| Series (1) |
| GSE75745 |
Microarray characterization of Vhl; Vhl, Hif1a; Vhl, Hif2a and Vhl, Hif1a, Hif2a deficient primary kidney cells |
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